The long term goal of the proposed research is to transform traditional cancer therapy protocols by including a pre-treatment step involving perturbing the vasculature of tumors with ultrasound- activated microbubbles (MBs). The new paradigm in cancer treatment protocol will result in significant increases in tumor kill using radiation, chemotherapy, hyperthermia or a combination of therapies. The technique further allows targeting of tumor specific volumes allowing healthy tissues to be spared because a lower dose of therapy can be applied. The American Cancer Society estimates that approximately 571,950 cancer deaths in America will occur in 2011 and about 1,596,670 new cases of cancer will occur in the U.S. The National Institutes of Health estimates overall costs of cancer in 2010 at $263.8 billion. However, these trends are improving because of continued developments in diagnostics and therapy. Therefore, continued improvements in these trends will depend on continued advancements in diagnostics and therapy. Our novel cancer therapy technique is based on using ultrasound-activated MBs in a way that will fundamentally change cancer therapy by amplifying the effects of treatment on cell death and spatially targeting therapy. We have recently discovered that ultrasound and MB-mediated cellular perturbation can enhance killing of cells in vitro from radiation or hyperthermia. Furthermore, we have demonstrated in animal models in vivo that ultrasound-activated MB perturbation of tumor vasculature results in significant enhancement in tumor kill when followed by traditional cancer therapy. Despite our emerging data on the ability of MBs to amplify therapeutic response of cells in vitro and in vivo, it is necessary to optimize this comprehensively, explore possible mechanisms responsible for these findings, and further test this phenomenon in vivo. The medical significance of adopting these techniques clinically is substantial because it could markedly improve existing therapies and reduce the side-effects associated with traditional therapies. Therefore, we propose a set of three specific aims to test, develop, optimize, demonstrate and quantify the efficacy of this novel technique. The first specific aim is to characterize the observed response by quantifying gene expression and cell signaling pathways relevant to therapy enhancement resulting from exposure to ultrasound-activated MBs. This specific aim (SA1) will be completed by conducting cell culture studies on cancer cell lines, conducting assays to examine cell signaling pathways associated with ultrasound-activated MB perturbation, and using animal models with ceramide deficient pathways. The second and third specific aims are to quantify, assess, and optimize the ability of ultrasound-activated MBs to synergistically enhance therapeutic response of tumors in vivo to radiation (SA 2), and to hyperthermia (SA 3). To meet these aims, several animal models of cancer will be exposed to the therapy techniques using different sets of exposure conditions and the efficacy of these therapies and exposure conditions assessed in vivo.